Conformational changes in the human Cx43/GJA1 gap junction channel visualized using cryo-EM

Connexin family proteins assemble into hexameric hemichannels in the cell membrane. The hemichannels dock together between two adjacent membranes to form gap junction intercellular channels (GJIChs). We report the cryo-electron microscopy structures of Cx43 GJICh, revealing the dynamic equilibrium state of various channel conformations in detergents and lipid nanodiscs. We identify three different N-terminal helix conformations of Cx43—gate-covering (GCN), pore-lining (PLN), and flexible intermediate (FIN)—that are randomly distributed in purified GJICh particles. The conformational equilibrium shifts to GCN by cholesteryl hemisuccinates and to PLN by C-terminal truncations and at varying pH. While GJIChs that mainly comprise GCN protomers are occluded by lipids, those containing conformationally heterogeneous protomers show markedly different pore sizes. We observe an α-to-π-helix transition in the first transmembrane helix, which creates a side opening to the membrane in the FIN and PLN conformations. This study provides basic structural information to understand the mechanisms of action and regulation of Cx43 GJICh.

Editorial Note: This manuscript has been previously reviewed at another journal that is not operating a transparent peer review scheme. This document only contains reviewer comments and rebuttal letters for versions considered at Nature Communications.

REVIEWERS' COMMENTS
Reviewer #1 (Remarks to the Author): Since the last version, the authors have made additional revisions to make it more comprehensible for readers.
The authors have addressed all my concerns.
I have no further comments.
Reviewer #2 (Remarks to the Author): The revised work by Lee et al. reflects a careful and attentive response to my previous critiques. I have no further significant concerns. The authors are congratulated on their efforts to characterize this important system! I do provide a couple minor comments below that the authors might consider in preparing a final submission, which should be readily addressed without further review.
Kind regards -

Minor
Line 250: "…indicating that the heads are more ordered than the tails." May be more appropriate to state "…indicating that the heads are more ordered than the tails and/or display enhanced electron scattering by the composition of heavier atoms." Line 259: "…, and thus the central pore formed by…" consider changing to the following for improved clarity "…, and thus the cytoplasmic end of the central pore formed by…" 1. It is not clear the pink cylinder representing the NT in Fig. 6C (FIN conformation). This representation is confusing since the other panels in Fig. 6C (for GCN and PLN) only show 1 NT instead of two and they are not represented as a cylinder. This is not explained in figure legend neither any part of the manuscript.
2. Related to the edited abstract: -Lines 38-39: "The conformational equilibrium shifted to GCN by cholesteryl hemisuccinates and to PLN by C-terminal truncations.". This statement is not fully accurate since PLN conformation was also observed without CT deletion (i.e., at PH=6.9).
-Lines 41-43: "We observed an α-to-π-helix transition in the first transmembrane helix, which created a side opening to the membrane in the FIN conformation." Opening was also observed for PLN conformation. Figure 3 what condition is shown.

Responses to Reviewers
We thank the reviewers for their time and effort to thoroughly read our point-by-point response and the revised manuscript and give us minor comments to increase the quality of the manuscript. All changes in the text have been highlighted by blue letters.

Minor points
(1) Line 250: "…indicating that the heads are more ordered than the tails." May be more appropriate to state "…indicating that the heads are more ordered than the tails and/or display enhanced electron scattering by the composition of heavier atoms." We agree with the reviewer and have modified the sentence according to the reviewer's suggestion (Line 248).
(2) Line 259: "…, and thus the central pore formed by…" consider changing to the following for improved clarity "…, and thus the cytoplasmic end of the central pore formed by…" We agree with the reviewer and have modified the sentence according to the reviewer's suggestion (Line 258).
(3) Line 274: Ideally, the authors would state what are the quantitative differences in [CHS] under these two conditions in the main text.
As suggested by the reviewer, we have added approximate concentrations (mol%) of CHS in the nanodisc samples under condition 7 and 8. The corresponding sentences have been modified as following.
"Because the protein sample included only 0.0005% (w/v) CHS, the lipid-protein mixture contained approximately 300 fold more phospholipid molecules than CHS molecules. In addition, CHS as well as LMNG would be partly removed during the incubation with adsorbent beads. Therefore, the CHS content in the lipid nanodisc sample was likely less than 0.3 mol%, which should be much lower than that under condition 7 (less than 50 mol%)." (Line 271-276)

Minor points
(1) It is not clear the pink cylinder representing the NT in Fig. 6C (FIN conformation). This representation is confusing since the other panels in Fig. 6C (for GCN and PLN) only show 1 NT instead of two and they are not represented as a cylinder. This is not explained in figure legend neither any part of the manuscript.
Following the reviewer's suggestion, we have added an appropriate explanation in the Figure 6C legend.
(2)-1 Lines 38-39: "The conformational equilibrium shifted to GCN by cholesteryl hemisuccinates and to PLN by C-terminal truncations.". This statement is not fully accurate since PLN conformation was also observed without CT deletion (i.e., at PH=6.9).
We agree with the reviewer and have modified the sentence as the editor suggested.
"The conformational equilibrium shifts to GCN by cholesteryl hemisuccinates and to PLN by C-terminal truncations and at varying pH." (Line 38) (2)-2 Lines 41-43: "We observed an α-to-π-helix transition in the first transmembrane helix, which created a side opening to the membrane in the FIN conformation." Opening was also observed for PLN conformation.
We agree with the reviewer and have added "the PLN conformation" in the sentence (Line 43). Figure 3 what condition is shown.

(3) Please specify in legend of
We have added the explanation in the Figure 3 legend.

(4) Lines 326-328 "However, this equilibrium state of Cx43-WT GJIChs in condition 3 would not represent the maximally open state of the channels at pH 6.9 in the previous electrophysiological experiments." Please incorporate the reference to the paper with electrophysiological experiments.
Following the reviewer's suggestion, we have referenced the paper.

(1) Details on P-and T-coupling (algorithm and coupling constant) are missing.
To control the pressure and temperature of systems, we used the Parrinello-Rahman (Ref. 62 in the manuscript) and the Nose-Hoover schemes (Ref. 63 in the manuscript), respectively. Along with the references, we explicitly specified those schemes in the revised manuscript.
Line 946: "at zero surface tension (γ = 0) 62 and 300 K temperature 63 " to "at zero surface tension (γ = 0) using the Parrinello-Rahman scheme 62 and 300 K temperature using the Nose-Hoover scheme 63 " (2) Are you sure that you constrained water molecules with LINCS, which is rather uncommon? The use of SETTLE is more common for water molecules with Gromacs.
Although we used the SETTLE algorithm for water molecules and correctly cited the original SETTLE paper (Ref. 66 in the manuscript), SETTLE was not explicitly stated in the original manuscript. We have added the following statement in the revised manuscript.
Line 951: "using the LINCS 65 and algorithms 66 , respectively." to "using the LINCS 65 and SETTLE 66 algorithms, respectively." (3) The Charmm-modified TIP3P water model (as the normal TIP3P model) exhibits a too large diffusion coefficient, which may lead to increased ionic currents relative to experiments. This limitation does not affect the main conclusions of this article but may affect the qualitative results of the simulations. The authors may either mention and discuss this limitation or carry out a control simulation with a different water model with correct diffusion coefficient (such as the OPC, TIP4P/2005, or TIP3P-force-balance). I would leave the decision to the authors, either solution is fine.
As pointed out by the reviewer, it is well-known that the TIP3P water model underestimates the viscosity by a factor of three (Ref. Lee et al.). As mentioned by the reviewer, we believe that this deviation does not affect the main conclusion of the manuscript because the effect of viscosity on current measurements can be removed by scaling the currents by a constant factor. As suggested by the reviewer, we have added the following sentence in Methods section.   Fig. 1d instead of Fig. 1c?
Yes. We have corrected the error.
(6) The Zenodo archive at https://zenodo.org/record/7219679 contains only structures and trajectories. For reproducibility, topologies, MD parameter (mdp), and (if applicable) index files should be added for all systems.